Method of producing partially purified extracellular metabolite products from Bacillus coagulans and biological applications thereof

ABSTRACT

Method of extracting a partially purified extracellular metabolite composition from  Bacillus coagulans  MTCC 5856 and its antimicrobial effects thereof are described.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

This patent application is the non-provisional filing for provisionalpatent application U.S. 61/920,567 filed 24 Dec. 2013 for the inventiontitled “METHOD OF PRODUCING PARTIALLY PURIFIED EXTRACELLULAR METABOLITEPRODUCTS FROM BACILLUS COAGULANS AND BIOLOGICAL APPLICATIONS THEREOF.”

BACKGROUND OF THE INVENTION

Field of the Invention

The invention in general relates to the anti-microbial effects ofprobiotic preparations. More specifically, the present invention relatesto (1) a method for producing partially purified extracellularmetabolite preparation from the probiotic bacterial strain Bacilluscoagulans SBC37-01 (Deposited in the Microbial Type Culture Collectionand Gene Bank and was assigned the strain number MTCC 5856) exhibiting99% genetic homology with the known bacterial strains Bacillus coagulansATCC 31284, Bacillus coagulans NBRC 3887 and Bacillus coagulans ATCC7050 and (2) the anti-microbial profile of said extracellular metabolitepreparation against a panel of microbial pathogens, includingsynergistic anti-microbial effects of preparation when combined with asynergistic preservative blend comprising from about 61% w/w of thymol,about 38% of monolaurin and about 1% w/w of magnolol obtained fromsupercritical fluid extracts of Magnolia officinalis. The extracellularmetabolite preparation alone or the combination of said extracellularmetabolite preparation and preservative blend is also shown to inhibitmicrobial biofilm formation in a synergistic manner.

Description of Prior Art

Extracellular products of Bacillus coagulans comprising a supernatant orfiltrate of a culture Bacillus coagulans strain suitable for topicalapplication to the skin or mucosal membranes of a mammal and therebycapable of being utilized to inhibit the growth of bacterium, yeast,fungi, virus, and combinations thereof is known in the art (U.S. Pat.No. 6,905,692, “Topical compositions containing probiotic Bacillusbacteria, spores, and extracellular products and uses thereof). Thepresent invention pertains to further purification of extracellularcomponents of cultures of probiotic Bacillus coagulans SBC37-01(Deposited in the Microbial Type Culture Collection and Gene Bank andwas assigned the strain number MTCC 5856) to obtain a concentratedextracellular metabolite preparation that exhibits enhancedanti-microbial effects when compared to the supernatant itself bothalone and when combined with a synergistic preservative blend comprisingfrom about 61% w/w of thymol, about 38% of monolaurin and about 1% w/wof magnolol obtained from supercritical fluid extracts of Magnoliaofficinalis.

It is the principle objective of the present invention to disclose

-   -   1) A method for producing partially purified extracellular        metabolite preparation from the probiotic bacterial strain        Bacillus coagulans SBC37-01 (Deposited in the Microbial Type        Culture Collection and Gene Bank and was assigned the strain        number MTCC 5856) exhibiting 99% genetic homology with the known        bacterial strains Bacillus coagulans ATCC 31284, Bacillus        coagulans NBRC 3887 and Bacillus coagulans ATCC 7050.    -   2) The anti-microbial profile of said extracellular metabolite        preparation against a panel of microbial pathogens, including        synergistic anti-microbial effects of preparation when combined        with a synergistic preservative blend comprising from about 61%        w/w of thymol, about 38% of monolaurin and about 1% w/w of        magnolol obtained from supercritical fluid extracts of Magnolia        officinalis.    -   3) The microbial biofilm inhibitory potential of said        extracellular metabolite preparation alone or in combination        with a synergistic preservative blend comprising from about 61%        w/w of thymol, about 38% of monolaurin and about 1% w/w of        magnolol obtained from supercritical fluid extracts of Magnolia        officinalis.

The present invention fulfills the aforesaid objectives and providesfurther related advantages.

DEPOSIT OF BIOLOGICAL MATERIAL

The deposit of biological material Bacillus coagulans SBC37-01 bearingaccession number MTCC 5856, mentioned in the instant application hasbeen made on 19 Sep. 2013 at Microbial Type Culture Collection & GeneBank (MTCC), CSIR-Institute of Microbial Technology, Sector 39-A,Chandigarh—160036, India.

SUMMARY OF THE INVENTION

The present invention describes

(1) A method for producing partially purified extracellular metabolitepreparation from the probiotic bacterial strain Bacillus coagulansSBC37-01 (Deposited in the Microbial Type Culture Collection and GeneBank and was assigned the strain number MTCC 5856) exhibiting 99%genetic homology with the known bacterial strains Bacillus coagulansATCC 31284, Bacillus coagulans NBRC 3887 and Bacillus coagulans ATCC7050;(2) The anti-microbial profile of said extracellular metabolitepreparation against a panel of microbial pathogens, includingsynergistic anti-microbial effects of preparation when combined with asynergistic preservative blend comprising from about 61% w/w of thymol,about 38% of monolaurin and about 1% w/w of magnolol obtained fromsupercritical fluid extracts of Magnolia officinalis and(3) The microbial biofilm inhibitory potential of said extracellularmetabolite preparation alone or in combination with a synergisticpreservative blend comprising from about 61% w/w of thymol, about 38% ofmonolaurin and about 1% w/w of magnolol obtained from supercriticalfluid extracts of Magnolia officinalis.(4) The antimicrobial activity and anti-acidogenic effect againstadherent S. mutans in water-insoluble glucans of said extracellularmetabolite preparation alone or in combination with a synergisticpreservative blend comprising from about 61% w/w of thymol, about 38% ofmonolaurin and about 1% w/w of magnolol obtained from supercriticalfluid extracts of Magnolia officinalis.

The present invention provides the following advantages.

-   -   1) Disclosure of a purification method for producing partially        purified extracellular metabolite preparation from the probiotic        bacterial strain Bacillus coagulans SBC37-01 (Deposited in the        Microbial Type Culture Collection and Gene Bank and was assigned        the strain number MTCC 5856) exhibiting 99% genetic homology        with the known bacterial strains Bacillus coagulans ATCC 31284,        Bacillus coagulans NBRC 3887 and Bacillus coagulans ATCC 7050;    -   2) Disclosure of the anti-microbial profile of the partially        purified against a panel of microbial pathogens, including        synergistic anti-microbial effects of said partially purified        extracellular preparation when combined with a synergistic        preservative blend comprising from about 61% w/w of thymol,        about 38% of monolaurin and about 1% w/w of magnolol obtained        from supercritical fluid extracts of Magnolia officinalis.    -   3) Disclosure of the microbial biofilm inhibitory potential of        said extracellular metabolite preparation alone or in        combination with a synergistic preservative blend comprising        from about 61% w/w of thymol, about 38% of monolaurin and about        1% w/w of magnolol obtained from supercritical fluid extracts of        Magnolia officinalis.    -   4) Disclosure of the antimicrobial activity and anti-acidogenic        effect against adherent S. mutans in water-insoluble glucans, of        said extracellular metabolite preparation alone or in        combination with a synergistic preservative blend comprising        from about 61% w/w of thymol, about 38% of monolaurin and about        1% w/w of magnolol obtained from supercritical fluid extracts of        Magnolia officinalis.

Other features and advantages of the present invention will becomeapparent from the following more detailed description, taken inconjunction with the accompanying images, which illustrate, by way ofexample, the principle of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the process flowchart for a purification method to producepartially purified extracellular metabolite preparation from theprobiotic bacterial strain Bacillus coagulans SBC37-01 (Deposited in theMicrobial Type Culture Collection and Gene Bank and was assigned thestrain number MTCC 5856) exhibiting 99% genetic homology with the knownbacterial strains Bacillus coagulans ATCC 31284, Bacillus coagulans NBRC3887 and Bacillus coagulans ATCC 7050.

FIG. 2 shows the representation of checkerboard broth micro-dilutionmethod for synergistic anti-microbial studies.

FIGS. 3A-3B, 3C-3D, 3E-3F, 3G-3H, 3I-3J, 3K-3L, 3M-3N, 3O-3P showrespectively, the graphical representation of the effect of naturalpreservative blend and extracellular metaboliltes of B. coagulans MTCC5856 alone and in combination on the growth and viability of Pseudomonasaeruginosa ATCC 9027, Escherichia coli ATCC 25922, Salmonella abony NCIM2257, Streptococcus mutans MTCC 1943, Propionibacterium acnes ATCC11827, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC14990 and Bacillus cereus ATCC 14579.

FIGS. 4A, 4B, 4C, 4D and 4E show respectively, the graphicalrepresentations of the effect of natural preservative blend andextracellular metabolites of B. coagulans MTCC 5856 alone and incombination on the biofilm formation of Pseudomonas aeruginosa ATCC9027, Escherichia coli ATCC 25922, Streptococcus mutans MTCC 1943,Staphylococcus aureus ATCC 29213 and Staphylococcus epidermidis ATCC14990.

FIGS. 5A and 5B show respectively, the graphical representations of theeffect of natural preservative blend and extracellular metabolites of B.coagulans MTCC 5856 on the growth/pH drop and the formation of waterinsoluble glucans of S. mutans MTCC 1943 biofilm in presence of 2%sucrose.

DETAILED DESCRIPTION OF THE MOST PREFERRED EMBODIMENT

In the most preferred embodiment the present invention relates to apurification method for producing partially purified extracellularmetabolite preparation from the probiotic bacterial strain Bacilluscoagulans SBC37-01 (Deposited in the Microbial Type Culture Collectionand Gene Bank and was assigned the strain number MTCC 5856) exhibiting99% genetic homology with the known bacterial strains Bacillus coagulansATCC 31284, Bacillus coagulans NBRC 3887 and Bacillus coagulans ATCC7050, said purification method comprising the steps of:

-   -   1. Inoculating a culture of Bacillus coagulans MTCC 5856        exhibiting 99% genetic homology with the known bacterial strains        Bacillus coagulans ATCC 31284, Bacillus coagulans NBRC 3887 and        Bacillus coagulans ATCC 7050 into 1.0 liter of Glucose Yeast        Extract Acetate broth medium (HiMedia, Mumbai India) or MRS        broth containing 0.5% tween 80 or Corn steep powder media;    -   2. Allowing the fermentation in the inoculated medium of step 1        to proceed for 24-48 h at 37° C. with 120 rpm;    -   3. Centrifuging the fermentation broth of step 2 at 4000-7000        rpm;    -   4. Concentrating supernatants 10 fold by using rotary evaporator        at 50° C. of step 3.    -   5. Adding 150 ml of chilled acetone drop by drop to 100 ml of        tenfold concentrated supernatants of step 4, followed by mixing;    -   6. Incubating the mixture of step 5 at 0° C. for 30 minutes        followed by centrifuging at 7000-8000 rpm;    -   7. Discarding the pellet obtained in step 6 and collecting 60%        acetone saturated supernatant (˜200 ml).    -   8. Concentrating the acetone saturated supernatant in step 7 to        50 ml by rotary evaporator.    -   9. Adjusting the pH to 5.0 by using 4N HCl, filtered (0.22        micron; Millex, Millipore, India) and stored at −20° C. till        further use.    -   10. Freeze drying/spray drying/tray drying the supernatant of        step 8.

In another most preferred embodiment, the present invention relates to aprocess of microbial control, said process comprising the step ofbringing into contact effective concentrations of a partially purifiedextracellular metabolite preparation from probiotic bacterial strainBacillus coagulans SBC37-01 (Deposited in the Microbial Type CultureCollection and Gene Bank and was assigned the strain number MTCC 5856)exhibiting 99% genetic homology with the known bacterial strainsBacillus coagulans ATCC 31284, Bacillus coagulans NBRC 3887 and Bacilluscoagulans ATCC 7050 and a target microbial cell. In more specificembodiments, the microbial cell may be one selected from the groupcomprising Pseudomonas aeruginosa, Escherichia coli, Staphylococcusaureus, Staphylococcus epidermidis, Streptococcus mutans,Propionibacterium acnes, Bacillus cereus and Salmonella abony.

In yet another most preferred embodiment, the present invention relatesto a process of microbial control, said process comprising the step ofbringing into contact a microbial cell with effective concentrations ofa preparation consisting essentially of partially purified extracellularmetabolite preparation from probiotic bacterial strain Bacilluscoagulans SBC37-01 (Deposited in the Microbial Type Culture Collectionand Gene Bank and was assigned the strain number MTCC 5856) exhibiting99% genetic homology with the known bacterial strains Bacillus coagulansATCC 31284, Bacillus coagulans NBRC 3887 and Bacillus coagulans ATCC7050 and a synergistic preservative blend comprising from about 61% w/wof thymol, about 38% of monolaurin and about 1% w/w of magnolol obtainedfrom supercritical fluid extracts of Magnolia officinalis. In morespecific embodiments, the microbial cell may be one selected from thegroup comprising Pseudomonas aeruginosa, Escherichia coli,Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans,Propionibacterium acnes, Bacillus cereus and Salmonella abony.

In yet another most preferred embodiment, the present invention alsorelates to a process of inhibiting microbial biofilm formation, saidprocess comprising step of bringing into contact biofilm producingmicrobial cells and a preparation consisting essentially of partiallypurified extracellular metabolite preparation from probiotic bacterialstrain Bacillus coagulans SBC37-01 (Deposited in the Microbial TypeCulture Collection and Gene Bank and was assigned the strain number MTCC5856) exhibiting 99% genetic homology with the known bacterial strainsBacillus coagulans ATCC 31284, Bacillus coagulans NBRC 3887 and Bacilluscoagulans ATCC 7050 alone or said preparation combined with asynergistic preservative blend comprising from about 61% w/w of thymol,about 38% of monolaurin and about 1% w/w of magnolol obtained fromsupercritical fluid extracts of Magnolia officinalis.

The following examples are presented herewith to illustrate theexemplary embodiments of the present invention.

Example 1 Microorganisms and Culture Conditions

The bacterial strains used in this study included Streptococcus mutansMTCC 1943, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidisATCC 14990, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC9027, Salmonella abony NCIM 2257 and Bacillus cereus ATCC 14579. Thereference strains were purchased from ATCC (American Type CultureCollection, Manassas, Va., USA), MTCC (IMTECH, Chandigarh, India) andNCIM (National Collection of Industrial Microorganisms, Pune, India). S.mutans and P. acnes were maintained on brain-heart infusion agar (BHI;Difco Laboratories, Detroit, Mich., USA) and reinforced clostridial agar(RCA; HiMedia, Mumbai, India) respectively. S. aureus, S. epidermidis E.coli, P. aeruginosa, S. abony and B. cereus were maintained ontrypticase soy agar (Difco Laboratories) at 37° C. S. mutans and P.acnes were incubated anaerobically (80% N₂, 10% H₂ and 10% CO₂) at 37°C. up to 48 h in anaerobic chamber (Coy Laboratory Products Inc,Michigan). Bacillus coagulans SBC37-01 used in the study wascharacterized and deposited to Microbial Type Culture Collection,Chandigarh, India and the strain was assigned as Bacillus coagulans MTCC5856.

Technique—The Checkerboard Method for Synergy Study

This is the most frequently used method to access the antimicrobialcombinations in vitro. The term “checkerboard” refers to the pattern (oftubes or microtiter plate wells) formed by multiple dilutions of twodrugs being tested (Eliopoulos G M, Moellering R C: Antimicrobialcombinations. In Antibiotics in laboratory medicine. Edited by Lorian V.Baltimore, The Williams & Wilkins Co; 1991:432-492). In the presentstudy, the checker board consisted of columns in which each tube (orwell) contains the same amount of the partially purified extracellularmetabolite preparation from the probiotic bacterial strain Bacilluscoagulans MTCC 5856 being diluted along the X-axis (rows) in which eachtube (or well) contains the same amount of the preservative blend beingdiluted on the Y-axis (FIG. 2). As a result each square in thecheckerboard (which represents one tube/well or plate) contained aunique combination of partially purified extracellular metabolitepreparation from the probiotic bacterial strain Bacillus coagulans MTCC5856 and preservative blend. The concentration range of preservativeblend in the present study was 2000 μg/ml to 31.25 μg/ml and lower insome cases, whereas the partially purified extracellular metabolitepreparations from the probiotic bacterial strain Bacillus coagulans MTCC5856 was tested in the range of 8% (v/v) to 0.015% (v/v). Thischeckerboard technique can be performed with liquid or semisolid (agar)media. BHI broth and RC broth were used for S. mutans and P. acnesrespectively and plates were incubated anaerobically (80% N₂, 10% H₂ and10% CO₂) at 37° C. up to 48 h in anaerobic chamber. Mueller hinton broth(Difco) was used for S. aureus, S epidermidis E. coli, P. aeruginosa, S.abony and B. cereus and plates were incubated at 37° C. for 18 h.

Kill Kinetics

Time-kill studies of the preservative blend and partially purifiedextracellular metabolite preparation from strain Bacillus coagulans MTCC5856 were conducted against P. aeruginosa, E. coli, S. aureus S.epidermidis, S. mutans, P. acnes, B. cereus and Salmonella abony andevaluated using a time-kill curve method, as described previously(Eliopoulus G M, Moellering R C J. Antimicrobial combinations. In:Lorian V, ed. Antibiotics in Laboratory Medicine, 4th edn. Baltimore:Williams & Wilkins, 1996; 330-6.). Bacterial suspension in itslogarithmic phase (1×10⁻⁶ cfu/ml) was used as the inoculum. Thepreservative blend and partially purified extracellular metabolitepreparation from strain Bacillus coagulans MTCC 5856 were tested aloneand in combination at different concentrations, as determined incheckerboard assay. The cell population as cfu/ml was determined by aserial dilution method in triplicate on respective media and incubatedin respective growth conditions. The viable count and absorbance (OD₆₁₀)were taken at 0 (untreated control), and 24 h of incubation at 37° C.Viable count was expressed in Log₁₀ cfu/ml and absorbance was expressedin OD value at 610 nm.

Biofilm Susceptibility Assays

The biofilm inhibitory effect of preservative blend and partiallypurified extracellular metabolite preparation from the strain Bacilluscoagulans MTCC 5856 alone and in combination were examined against S.mutans, E. coli, S. aureus S. epidermidis and P. aerugenosa by themicrodilution method (Wei et al. Journal of Antimicrob. Chemother. 2006.57:1100-1109.). This method was similar to the checkerboard method forplanktonic cells. For S. mutans biofilm assay, BHI broth supplementedwith 2% sucrose was used. In case of other organisms, TSB supplementedwith 2% glucose was used in the study. The bacterial suspensions wereprepared from the overnight-grown culture, and the turbidity of thesuspension was adjusted to an optical density at 610 nm (A₆₁₀) of 0.7(1×10⁹ CFU/ml). The concentration range of preservative blend in thepresent study was 2000 μg/ml to 31.25 μg/ml and lower concentrations insome cases, whereas partially purified extracellular metabolitepreparation from the strain Bacillus coagulans MTCC 5856 was tested inthe range of 8% (v/v) to 0.015% (v/v). Forty microliters of fresh mediabroth was added to each well, followed by the addition of 60 μl of theabove-mentioned suspension to each well of the plate. This resulted inthe final inoculum of 6×10⁷ CFU/ml in each well; After incubation at 37°C. for 48 h, the culture supernatant from each well was decanted, andplanktonic cells were removed by washing the wells withphosphate-buffered saline (PBS; pH 7.2). The biofilm was fixed withmethanol for 15 min and then air dried at room temperature. The wells ofthe dried plate were stained with 0.1% (w/v) crystal violet (SigmaChemical Co., St Louis, Mo.) for 10 min and rinsed thoroughly with wateruntil the negative control wells appeared colorless. Biofilm formationwas quantified by the addition of 200 μl of 95% ethanol to the crystalviolet-stained wells and recording the absorbance at 595 nm (A₅₉₅). Thepercentage of biofilm inhibition was calculated using the equation (A₅₉₅of biofilm treated with test agent/A₅₉₅ of non-treated control)×100.Culture without test agent was used as the non-treated control.

Antimicrobial Activity Against Adherent S. mutans in Water-InsolubleGlucan

The formation of water-insoluble glucan by S. mutans MTCC 1943 wasperformed by a previously described method (Katsura et al., Antimicrob.Agents Chemother. 2001. 45:3009-3013). Briefly, aliquots of 100 μl ofculture of S. mutans MTCC 1943 (—1×10⁸ cells/ml) were inoculated into 10ml of fresh BHI broth containing 2% sucrose (w/v) in the test tubes andincubated at 37° C. for 24 h at an inclination of 300. The fluidcontaining planktonic cells was gently removed. The water-insolubleglucan containing cells of S. mutans MTCC 1943 were gently washed with10 ml of sterile water and resuspended in 10 ml of phosphate buffer (10mM, pH 5.0) containing preservative blend and partially purifiedextracellular metabolite preparation from the strain Bacillus coagulansMTCC 5856 alone and in combination, followed by incubation at 37° C. for5 min. Chlorhexidine 0.12% (v/v) (Sigma Chemical Co., St Louis, Mo.) wasused as internal reference in the study. The mixture was gently washedagain with sterile saline (0.89% NaCl, w/v), followed by theresuspension of treated cells in 10 ml of BHI broth containing 2%sucrose (w/v). After incubation of cells at 37° C. for 6, 12, 18, and 24h, the acid produced by the culture was measured by using a pH meter.The fluid containing free cells of S. mutans MTCC was gently removed.The water insoluble glucan was resuspended in 10 ml of 1 N NaOH solutionand homogenized; the turbidity was measured at 610 nm.

RESULTS

Table 1 shows the fold reduction of human microbial pathogens whenbrought in contact with A. formulations containing just PartiallyPurified Extracellular Metabolites (PPEM) or Natural Preservative Blend(NPB); and B. formulations incorporating both Partially PurifiedExtracellular Metabolites (PPEM) and Natural Preservative Blend (NPB).It may be noted that the formulations incorporating both PartiallyPurified Extracellular Metabolites (PPEM) and Natural Preservative Blend(NPB) cause a significant eight fold decrease for pathogen Pseudomonasaeruginosa and four fold decrease for pathogen Escherichia coli.

TABLE 1 Results of checkerboard testing of the natural preservativeblend and partially purified extracellular metabolites of probioticstain Bacillus coagulans MTCC 5856 against human pathogens PartiallyPurified Extracellular Metabolites (PPEM) Natural Preservative Blend(NPB) MIC (%, v/v) MIC (μg/ml) In combination with NPB In combinationwith PPEM S. No Tested organisms alone (Fold reduction in MIC) alone(Fold reduction in MIC) 1 P. aeruginosa ATCC 9027 0.5 0.12 (4) 250 31.25(8) 2 E. coli ATCC 25922 1.0 0.25 (4) 250 62.50 (4) 3 S. abony NCIM 22571.0 0.25 (4) 500 125 (4) 4 S. mutans MTCC 1943 4.0 2.0 (2) 15.62 7.81(2) 5 P. acnes ATCC 11827 4.0 2.0 (2) 62.50 31.25 (2) 5 S. aureus ATCC29213 2.0 1.0 (2) 31.25 15.62 (2) 6 S. epidermidis ATCC 14990 2.0 1.0(2) 32.25 15.62 (2) 7 B. cereus ATCC 14579 4.0 2.0 (2) 125 31.25 (2)

While the invention has been described with reference to a preferredembodiment, it is to be clearly understood by those skilled in the artthat the invention is not limited thereto. Rather, the scope of theinvention is to be interpreted only in conjunction with the appendedclaims.

We claim:
 1. A purification method for producing partially purifiedextracellular metabolite preparation from the probiotic bacterial strainBacillus coagulans strain MTCC 5856), said purification methodcomprising the steps of: a) Inoculating a culture of Bacillus coagulansMTCC 5856 into 1.0 liter of Glucose Yeast Extract Acetate broth mediumor MRS broth containing 0.5% Tween 80 or Corn steep powder media toinitiate bacterial fermentation; b) Allowing the bacterial fermentationin the inoculated medium of step a to proceed for 24-48 h at 37° C. with120 rpm; c) Centrifuging the fermentation broth of step b at 4000-7000rpm and collecting supernatant; d) Concentrating supernatants 10 fold byusing rotary evaporator at 50° C. of step c; e) Adding 150 ml of chilledacetone drop by drop to 100 ml of tenfold concentrated supernatants ofstep d, followed by mixing to form supernatants-acetone mixture; f)Incubating the supernatants-acetone mixture of step e at 0° C. for 30minutes followed by centrifuging at 7000-8000 rpm to collect a pellet;g) Discarding the pellet obtained in step f and collecting 60% (v/v)acetone saturated supernatant (˜200 ml); h) Concentrating the acetonesaturated supernatant in step g to 50 ml by rotary evaporator; i)Adjusting the pH of the supernatant of step h to 5.0 by using 4N HCl,filtered (0.22 micron; Millex, Millipore, India) and stored at −20° C.till further use; j) Freeze drying the supernatant of step I to obtainpartially purified extracellular metabolite preparation from theprobiotic bacterial strain Bacillus coagulans MTCC 5856.